Methods, compositions, and kits for the treatment of ophthalmic disorders

ABSTRACT

The invention features methods, kits, and compositions for the treatment of ophthalmic disorders. The compositions include a corticosteroid in combination with a non-steroidal immunophilin-dependent immunosuppressant.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 11/594,428, filed Nov. 8, 2006, which claims benefit of U.S.Provisional Application No. 60/735,989, filed Nov. 9, 2005, each ofwhich is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

The invention relates to the treatment of ophthalmic disorders.

Dry eye, also known generically as keratoconjunctivitis sicca, is acommon ophthalmological disorder affecting millions of Americans eachyear. The condition is particularly widespread among post-menopausalwomen due to hormonal changes following the cessation of fertility. Dryeye may afflict an individual with varying severity. In mild cases, apatient may experience burning, a feeling of dryness, and persistentirritation such as is often caused by small bodies lodging between theeye lid and the eye surface. In severe cases, vision may besubstantially impaired. Other diseases, such as Sjogren's disease andcicatricial pemphigoid, manifest dry eye complications.

Practitioners have taken several approaches to the treatment of dry eye.One common approach has been to supplement and stabilize the ocular tearfilm using so-called artificial tears instilled throughout the day.Other approaches include the use of ocular inserts that provide a tearsubstitute or stimulation of endogenous tear production.

SUMMARY OF THE INVENTION

The invention features compositions, methods, and kits for the treatmentof ophthalmic disorders.

In one aspect, the invention features a method of treating an ophthalmicdisorder in a patient by administering to the patient a corticosteroidand a non-steroidal immunophilin-dependent immunosuppressant (NsIDI). Inthis aspect of the invention, the corticosteroid and/or thenon-steroidal immunophilin-dependent immunosuppressant can beadministered at a low concentration. Desirably, the concentration of thenon-steroidal immunophilin-dependent immunosuppressant does not causeeye irritation, such as burning, and the compositions of the inventionare administered in an amount sufficient to alleviate the symptoms ofthe ophthalmic disorder. Also desirably, the concentration of thecorticosteroid does not cause steroid toxicity.

In another aspect, the invention features a method of treating anophthalmic disorder in a patient by administering to the patient asubstance selected from: dipivefrin, anti-VEGF therapies, photodynamictherapy, NSAIDS, antiallergic agents, antihistamines, glaucoma-treatingagents, artificial tears, antibiotics, antiviral agents, and antimycoticagents in combination with a corticosteroid and/or an NsIDI. In thisaspect of the invention, the corticosteroid and/or the non-steroidalimmunophilin-dependent immunosuppressant can be administered at a lowconcentration.

In another aspect, the invention features a composition (e.g., asolution, gel, ointment, suspension, emulsion, or solid insert)including a corticosteroid and a NsIDI. In this aspect of the invention,the corticosteroid and/or the NsIDI can be administered at a lowconcentration.

In another aspect, the invention features a composition (e.g., asolution, gel, ointment, suspension, emulsion, or solid insert)including a substance selected from: dipivefrin, anti-VEGF therapies,photodynamic therapy, NSAIDS, antiallergic agents, antihistamines,glaucoma-treating agents, artificial tears, antibiotics, antiviralagents, and antimycotic agents in combination with a corticosteroidand/or an NsIDI. In this aspect of the invention, the corticosteroidand/or the non-steroidal immunophilin-dependent immunosuppressant can beadministered at a low concentration.

The invention also features a kit that includes (i) a corticosteroid;and (ii) instructions for administering a corticosteroid and an NsIDI toa patient having or at risk of having an ophthalmic disorder.

The invention also features a kit that includes (i) an NsIDI; and (ii)instructions for administering a corticosteroid and an NsIDI to apatient having or at risk of having an ophthalmic disorder.

The invention also features a kit that includes (i) a compositioncontaining a corticosteroid and an NsIDI; and (ii) instructions foradministering the composition to a patient having or at risk of having ametabolic disorder.

The invention also features a kit that includes (i) a corticosteroid;(ii) an NsIDI; and (iii) instructions for administering a corticosteroidand an NsIDI to a patient having or at risk of having an ophthalmicdisorder.

Any of the foregoing kits can also include instructions foradministering a compound selected from: dipivefrin, anti-VEGF therapies,photodynamic therapy, NSAIDS, antiallergic agents, antihistamines,glaucoma-treating agents, artificial tears, antibiotics, antiviralagents, and antimycotic agents.

Any of the foregoing kits can also include a compound selected from:dipivefrin, anti-VEGF therapies, photodynamic therapy, NSAIDS,antiallergic agents, antihistamines, glaucoma-treating agents,artificial tears, antibiotics, antiviral agents, and antimycotic agents.In these kits, the NsIDI and/or corticosteroid can optionally beformulated in a single composition with a compound selected from:dipivefrin, anti-VEGF therapies, photodynamic therapy, NSAIDS,antiallergic agents, antihistamines, glaucoma-treating agents,artificial tears, antibiotics, antiviral agents, and antimycotic agents.

The invention also features a kit that includes (i) a corticosteroid;and (ii) instructions for administering a corticosteroid and a compoundselected from: dipivefrin, anti-VEGF therapies, photodynamic therapy,NSAIDS, antiallergic agents, antihistamines, glaucoma-treating agents,artificial tears, antibiotics, antiviral agents, and antimycotic agents,to a patient having or at risk of having an ophthalmic disorder.

The invention also features a kit that includes (i) an NsIDI; and (ii)instructions for administering an NsIDI and a compound selected from:dipivefrin, anti-VEGF therapies, photodynamic therapy, NSAIDS,antiallergic agents, antihistamines, glaucoma-treating agents,artificial tears, antibiotics, antiviral agents, and antimycotic agents,to a patient having or at risk of having an ophthalmic disorder.

The invention also features a kit that includes (i) a compositioncontaining a corticosteroid and a compound selected from: dipivefrin,anti-VEGF therapies, photodynamic therapy, NSAIDS, antiallergic agents,antihistamines, glaucoma-treating agents, artificial tears, antibiotics,antiviral agents, and antimycotic agents; and (ii) instructions foradministering the composition to a patient having or at risk of having ametabolic disorder.

The invention also features a kit that includes (i) a compositioncontaining an NsIDI and a compound selected from: dipivefrin, anti-VEGFtherapies, photodynamic therapy, NSAIDS, antiallergic agents,antihistamines, glaucoma-treating agents, artificial tears, antibiotics,antiviral agents, and antimycotic agents; and (ii) instructions foradministering the composition to a patient having or at risk of having ametabolic disorder.

The invention also features a kit that includes (i) a corticosteroid;(ii) a compound selected from: dipivefrin, anti-VEGF therapies,photodynamic therapy, NSAIDS, antiallergic agents, antihistamines,glaucoma-treating agents, artificial tears, antibiotics, antiviralagents, and antimycotic agents (iii) instructions for administering acorticosteroid and a compound selected from: dipivefrin, anti-VEGFtherapies, photodynamic therapy, NSAIDS, antiallergic agents,antihistamines, glaucoma-treating agents, artificial tears, antibiotics,antiviral agents, and antimycotic agents, to a patient having or at riskof having an ophthalmic disorder.

The invention also features a kit that includes (i) a NsIDI; (ii) acompound selected from: dipivefrin, anti-VEGF therapies, photodynamictherapy, NSAIDS, antiallergic agents, antihistamines, glaucoma-treatingagents, artificial tears, antibiotics, antiviral agents, and antimycoticagents (iii) instructions for administering a NsIDI and a compoundselected from: dipivefrin, anti-VEGF therapies, photodynamic therapy,NSAIDS, antiallergic agents, antihistamines, glaucoma-treating agents,artificial tears, antibiotics, antiviral agents, and antimycotic agents,to a patient having or at risk of having an ophthalmic disorder.

In any of the forgoing aspects of the invention, the corticosteroid canbe selected from SEGRAs (selective glucocorticosteroid receptoragonists), fluocinolone acetonide, fluorometholone, dexamethasone,hydrocortisone, loteprednol, medrysone, methylprednisolone,prednisolone, rimexolone, or triamcinolone.

In any of the forgoing aspects of the invention, the NsIDI can beselected from cyclosporine A, ABT-281, ISAtx247, tacrolimus, ascomycin,pimecrolimus, rapamycin, or everolimus.

In any of the foregoing aspects of the invention, the concentration ofthe corticosteroid can be equivalent to a concentration of prednisoloneof between 0.01% and 0.1% (e.g., 0.1%, 0.09%, 0.08%, 0.07%, 0.06%,0.05%, or 0.01%) and the concentration of the non-steroidalimmunophilin-dependent immunosuppressant can be equivalent to aconcentration of cyclosporine A between 0.001% and 0.049% (e.g., 0.04%,0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, or 0.001%).

In one embodiment, the corticosteroid is prednisolone and theconcentration of prednisolone is between 0.01% and 0.12% (e.g., 0.12%,0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, and 0.01%).

In another embodiment, the corticosteroid is clocortolone pivalate andthe concentration of clocortolone pivalate is between 0.01% and 0.1%(e.g., 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, and 0.01%).

In another embodiment, the corticosteroid is hydrocortisone and theconcentration of hydrocortisone is between 0.01% and 1.0% (e.g., 1.0%,0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%,and 0.01%).

In another embodiment, the corticosteroid is dexamethasone and theconcentration of dexamethasone is between 0.01% and 0.1% (e.g., 0.1%,0.09%, 0.08%, 0.07%, 0.06%, 0.05%, and 0.01%).

In another embodiment, the corticosteroid is fluorometholone and theconcentration of fluorometholone is between 0.01% and 0.1% (e.g., 0.1%,0.09%, 0.08%, 0.07%, 0.06%, 0.05%, and 0.01%).

In another embodiment, the corticosteroid is loteprednol etabonate andthe concentration of loteprednol etabonate is between 0.01% and 0.2%(e.g., 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, and 0.01%).

In another embodiment, the corticosteroid is medrysone and theconcentration of medrysone is between 0.01% and 1.0% (e.g., 1.0%, 0.9%,0.8%, 0.7%, 0.6%, 0.5%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, and0.01%).

In another embodiment, the corticosteroid is rimexolone and theconcentration of rimexolone is between 0.01% and 1.0% (e.g., 1.0%, 0.9%,0.8%, 0.7%, 0.6%, 0.5%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, and0.01%).

In any of the foregoing embodiments, the non-steroidalimmunophilin-dependent immunosuppressant is cyclosporine A and theconcentration of cyclosporine A is between 0.001% and 0.049% (e.g.,0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, and0.001%).

In certain embodiments of the compositions, kits, and methods of theinvention, the only pharmacologically active agents in the compositionor kit, or used in the method, are those recited. In this embodiment,pharmacologically inactive excipients may also be present in thecomposition or kit, or used in the practice of the method.

The invention features the treatment of an ophthalmic disorder, forexample age related macular degeneration, alkaline erosivekeratoconjunctivitis, allergic conjunctivitis, allergic keratitis,anterior uveitis, Behcet's disease, blepharitis, blood-aqueous barrierdisruption, chorioiditis, chronic uveitis, conjunctivitis, contactlens-induced keratoconjunctivitis, corneal abrasion, corneal trauma,corneal ulcer, crystalline retinopathy, cystoid macular edema,dacryocystitis, diabetic keratophathy, diabetic macular edema, diabeticretinopathy, dry eye disease, dry age-related macular degeneration,eosinophilic granuloma, episcleritis, exudative macular edema, Fuchs'Dystrophy, giant cell arteritis, giant papillary conjunctivitis,glaucoma, glaucoma surgery failure, graft rejection, herpes zoster,inflammation after cataract surgery, iridocorneal endothelial syndrome,iritis, keratoconjunctiva sicca, keratoconjunctival inflammatorydisease, keratoconus, lattice dystrophy, map-dot-fingerprint dystrophy,necrotic keratitis, neovascular diseases involving the retina, uvealtract or cornea such as neovascular glaucoma, cornealneovascularization, neovascularization resulting following a combinedvitrectomy and lensectomy, neovascularization of the optic nerve, andneovascularization due to penetration of the eye or contusive ocularinjury, neuroparalytic keratitis, non-infectious uveitisocular herpes,ocular lymphoma, ocular rosacea, ophthalmic infections, ophthalmicpemphigoid, optic neuritis, panuveitis, papillitis, pars planitis,persistent macular edema, phacoanaphylaxis, posterior uveitis,post-operative inflammation, proliferative diabetic retinopathy,proliferative sickle cell retinopathy, proliferative vitreoretinopathy,retinal artery occlusion, retinal detachment, retinal vein occlusion,retinitis pigmentosa, retinopathy of prematurity, rubeosis iritis,scleritis, Stevens-Johnson syndrome, sympathetic ophthalmia, temporalarteritis, thyroid associated ophthalmopathy, uveitis, vernalconjunctivitis, vitamin A insufficiency-induced keratomalacia,vitreitis, or wet age-related macular degeneration.

Compounds useful in the invention include those described herein in anyof their pharmaceutically acceptable forms, including isomers such asdiastereomers and enantiomers, salts, esters, solvates, and polymorphsthereof, as well as racemic mixtures and pure isomers of the compoundsdescribed herein.

By “corticosteroid” is meant any naturally occurring or syntheticcompound characterized by a hydrogenatedcyclopentanoperhydro-phenanthrene ring system and havingimmunosuppressive and/or anti-inflammatory activity. Naturally occurringcorticosteroids are generally produced by the adrenal cortex. Syntheticcorticosteroids may be halogenated. Examples corticosteroids areprovided herein.

By “non-steroidal immunophilin-dependent immunosuppressant” or “NsIDI”is meant any non-steroidal agent that decreases proinflammatory cytokineproduction or secretion, binds an immunophilin, or causes a downregulation of the proinflammatory reaction. NsIDIs include calcineurininhibitors, such as cyclosporine A, ABT-281, ISAtx247, tacrolimus,ascomycin, pimecrolimus, as well as other agents (peptides, peptidefragments, chemically modified peptides, or peptide mimetics) thatinhibit the phosphatase activity of calcineurin. NsIDIs also includerapamycin (sirolimus) and everolimus, which bind to an FK506-bindingprotein, FKBP-12, and block antigen-induced proliferation of white bloodcells and cytokine secretion.

By a “low dosage” or “low concentration” is meant at least 5% less(e.g., at least 10%, 20%, 50%, 80%, 90%, or even 95%) than the loweststandard recommended dosage or lowest standard recommended concentration(e.g., less than the concentration approved by the FDA for ophthalmicadministration, see Table 1) of a particular compound formulated for agiven route of administration for treatment of any human disease orcondition. For example, a low dosage of corticosteroid formulated foradministration by inhalation will differ from a low dosage ofcorticosteroid formulated for oral administration of a particularcompound formulated for a given route of administration for treatment ofany human disease or condition.

By “treating” is meant administering or prescribing a pharmaceuticalcomposition for the treatment or prevention of an immunoinflammatorydisease.

By “patient” is meant any animal (e.g., a human). Other animals that canbe treated using the methods, compositions, and kits of the inventioninclude horses, dogs, cats, pigs, goats, rabbits, hamsters, monkeys,guinea pigs, rats, mice, lizards, snakes, sheep, cattle, fish, andbirds.

By a “concentration equivalent to a concentration of prednisolone” ismeant a concentration of a corticosteroid that produces the sameanti-inflammatory effect in a patient as a concentration ofprednisolone.

By a “concentration equivalent to a concentration of cyclosporine A” ismeant a concentration of a NsIDI that produces the sameanti-inflammatory effect in a patient as a concentration of cyclosporineA.

“Ophthalmic disorder” refers to physiologic abnormalities of the eye.They may involve the retina, the vitreous humor, lens, cornea, sclera orother portions of the eye, or physiologic abnormalities that adverselyaffect the eye, such as inadequate tear production, allergicconjunctivitis, uveitis or corneal transplant.

By “steroid toxicity” is meant a detrimental increase in intraocularpressure resulting from steroid administration.

Ophthalmic disorders that can be treated using the compositions,methods, and kits of the invention include age related maculardegeneration; alkaline erosive keratoconjunctivitis; allergicconjunctivitis; allergic keratitis; anterior uveitis (iridocyclitis);Behcet's disease; blepharitis; blood-aqueous barrier disruption;chorioiditis; chronic uveitis; conjunctivitis; contact lens-inducedkeratoconjunctivitis; corneal abrasion; corneal trauma; corneal ulcer(e.g., Mooren's ulcer, corneal ulcer subsequent to chronic rheumatoidarthritis or collagen disease, Terrien' margine degeneration, catarrhalcorneal ulcer, infectious corneal ulcer); crystalline retinopathy;cystoid macular edema; dacryocystitis; diabetic keratophathy; diabeticmacular edema; diabetic retinopathy; dry eye disease; dry age-relatedmacular degeneration; eosinophilic granuloma; episcleritis; exudativemacular edema; Fuchs' Dystrophy; giant cell arteritis; giant papillaryconjunctivitis; glaucoma; glaucoma surgery failure; graft rejection;herpes zoster (shingles); inflammation after cataract surgery;iridocorneal endothelial syndrome; iritis; keratoconjunctiva sicca;keratoconjunctival inflammatory disease; keratoconus; lattice dystrophy;map-dot-fingerprint dystrophy; necrotic keratitis; neovascular diseasesinvolving the retina, uveal tract or cornea such as neovascularglaucoma, corneal neovascularization (inflammatory, transplantation,developmental hypoplasia of the iris), neovascularization resultingfollowing a combined vitrectomy and lensectomy, neovascularization ofthe optic nerve, and neovascularization due to penetration of the eye orcontusive ocular injury; neuroparalytic keratitis; non-infectiousuveitisocular herpes; ocular lymphoma; ocular rosacea; ophthalmicinfections (e.g., corneal herpes, bacterial keratitis, bacterialconjunctivitis, mycotic keratitis, acanthamebic keratitis, infectiousendophthalmitis, infectious corneal ulcer, inflammation of theconjunctiva or cornea by staphylococci, streptococci, enterococci,euterococci, bacillus, corynebacterium, chlamydia, and neisseria);ophthalmic pemphigoid; optic neuritis; panuveitis; papillitis; parsplanitis; persistent macular edema; phacoanaphylaxis; posterior uveitis(chorioentinitis); post-operative inflammation; proliferative diabeticretinopathy; proliferative sickle cell retinopathy; proliferativevitreoretinopathy; retinal artery occlusion; retinal detachment; retinalvein occlusion; retinitis pigmentosa; retinopathy of prematurity;rubeosis iritis; scleritis; Stevens-Johnson syndrome (erythemamultiforme major); sympathetic ophthalmia; temporal arteritis; thyroidassociated ophthalmopathy (Graves' Ophthalmopathy); uveitis; vernalconjunctivitis; vitamin A insufficiency-induced keratomalacia;vitreitis; and wet age-related macular degeneration.

The term “pharmaceutically acceptable salt” represents those salts whichare, within the scope of sound medical judgment, suitable for use incontact with the tissues of humans and lower animals without unduetoxicity, irritation, allergic response and the like, and arecommensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art. The salts can be prepared insitu during the final isolation and purification of the compounds of theinvention, or separately by reacting the free base function with asuitable organic acid. Representative acid addition salts includeacetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate,benzoate, bisulfate, borate, butyrate, camphorate, camphersulfonate,citrate, cyclopentanepropionate, digluconate, dodecylsulfate,ethanesulfonate, fumarate, glucoheptonate, glycerophosphate,hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride,hydroiodide, 2-hydroxy-ethanesulfonate, isethionate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate, mesylate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts,and the like. Representative alkali or alkaline earth metal saltsinclude sodium, lithium, potassium, calcium, magnesium, and the like, aswell as nontoxic ammonium, quaternary ammonium, and amine cations,including, but not limited to ammonium, tetramethylammonium,tetraethylammonium, methylamine, dimethylamine, trimethylamine,triethylamine, ethylamine, and the like.

Compounds useful in the invention include those described herein in anyof their pharmaceutically acceptable forms, including isomers such asdiastereomers and enantiomers, salts, esters, amides, thioesters,solvates, and polymorphs thereof, as well as racemic mixtures and pureisomers of the compounds described herein.

Other features and advantages of the invention will be apparent from thefollowing detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1LL are graphs showing suppression of IFNγ, IL-2, and TNFα incells treated with combinations of an NsIDI and a corticosteroid.

DETAILED DESCRIPTION

The invention features methods, compositions, and kits for the treatmentof ophthalmic disorders. In one embodiment, a patient with an ophthalmicdisorder is treated by administering two drugs simultaneously.

Cyclosporine A, a non-steroidal immunophilin-dependent immunosuppressant(NsIDI), is approved for treating several ophthalmic conditions.Cyclosporine A causes eye irritation and other undesired side effectswhen administered to patients at the lowest approved concentration.Lower concentrations of cyclosporine A do not cause these undesired sideeffects but are not sufficient to alleviate the symptoms of theophthalmic disorders.

Both corticosteroids and NsIDIs suppress cytokine production in cellculture models of immune function. We have discovered that combinationsof certain NsIDIs with certain corticosteroids suppress cytokineproduction in a synergistic manner.

Based upon these data, we propose that when combined with acorticosteroid, low concentrations of cyclosporine A are sufficient toalleviate the symptoms of ophthalmic disorders while not causingundesired side effects.

Corticosteroids

In certain embodiments, a corticosteroid may be employed in a method,composition, or kit of the invention. Suitable corticosteroids includethose from the class of selective glucocorticosteroid receptor agonists(SEGRAs), 11-alpha,17-alpha,21-trihydroxypregn-4-ene-3,20-dione;11-beta,16-alpha,17,21-tetrahydroxypregn-4-ene-3,20-dione;11-beta,16-alpha,17,21-tetrahydroxypregn-1,4-diene-3,20-dione;11-beta,17-alpha,21-trihydroxy-6-alpha-methylpregn-4-ene-3,20-dione;11-dehydrocorticosterone; 11-deoxycortisol;11-hydroxy-1,4-androstadiene-3,17-dione; 11-ketotestosterone;14-hydroxyandrost-4-ene-3,6,17-trione; 15,17-dihydroxyprogesterone;16-methylhydrocortisone;17,21-dihydroxy-16-alpha-methylpregna-1,4,9(11)-triene-3,20-dione;17-alpha-hydroxypregn-4-ene-3,20-dione; 17-alpha-hydroxypregnenolone;17-hydroxy-16-beta-methyl-5-beta-pregn-9(11)-ene-3,20-dione;17-hydroxy-4,6,8(14)-pregnatriene-3,20-dione;17-hydroxypregna-4,9(11)-diene-3,20-dione; 18-hydroxycorticosterone;18-hydroxycortisone; 18-oxocortisol; 21-acetoxypregnenolone;21-deoxyaldosterone; 21-deoxycortisone; 2-deoxyecdysone;2-methylcortisone; 3-dehydroecdysone; 4-pregnene-17-alpha,20-beta,21-triol-3,11-dione; 6,17,20-trihydroxypregn-4-ene-3-one;6-alpha-hydroxycortisol; 6-alpha-fluoroprednisolone,6-alpha-methylprednisolone, 6-alpha-methylprednisolone 21-acetate,6-alpha-methylprednisolone 21-hemisuccinate sodium salt,6-beta-hydroxycortisol, 6-alpha, 9-alpha-difluoroprednisolone 21-acetate17-butyrate, 6-hydroxycorticosterone; 6-hydroxydexamethasone;6-hydroxyprednisolone; 9-fluorocortisone; alclomethasone dipropionate;aldosterone; algestone; alphaderm; amadinone; amcinonide; anagestone;androstenedione; anecortave acetate; beclomethasone; beclomethasonedipropionate; betamethasone 17-valerate; betamethasone sodium acetate;betamethasone sodium phosphate; betamethasone valerate; bolasterone;budesonide; calusterone; chlormadinone; chloroprednisone;chloroprednisone acetate; cholesterol; ciclesonide; clobetasol;clobetasol propionate; clobetasone; clocortolone; clocortolone pivalate;clogestone; cloprednol; corticosterone; cortisol; cortisol acetate;cortisol butyrate; cortisol cypionate; cortisol octanoate; cortisolsodium phosphate; cortisol sodium succinate; cortisol valerate;cortisone; cortisone acetate; cortivazol; cortodoxone; daturaolone;deflazacort, 21-deoxycortisol, dehydroepiandrosterone; delmadinone;deoxycorticosterone; deprodone; descinolone; desonide; desoximethasone;dexafen; dexamethasone; dexamethasone 21-acetate; dexamethasone acetate;dexamethasone sodium phosphate; dichlorisone; diflorasone; diflorasonediacetate; diflucortolone; difluprednate; dihydroelatericin a;domoprednate; doxibetasol; ecdysone; ecdysterone; emoxolone; endrysone;enoxolone; fluazacort; flucinolone; flucloronide; fludrocortisone;fludrocortisone acetate; flugestone; flumethasone; flumethasonepivalate; flumoxonide; flunisolide; fluocinolone; fluocinoloneacetonide; fluocinonide; fluocortin butyl; 9-fluorocortisone;fluocortolone; fluorohydroxyandrostenedione; fluorometholone;fluorometholone acetate; fluoxymesterone; fluperolone acetate;fluprednidene; fluprednisolone; flurandrenolide; fluticasone;fluticasone propionate; formebolone; formestane; formocortal;gestonorone; glyderinine; halcinonide; halobetasol propionate;halometasone; halopredone; haloprogesterone; hydrocortamate;hydrocortiosone cypionate; hydrocortisone; hydrocortisone 21-butyrate;hydrocortisone aceponate; hydrocortisone acetate; hydrocortisonebuteprate; hydrocortisone butyrate; hydrocortisone cypionate;hydrocortisone hemisuccinate; hydrocortisone probutate; hydrocortisonesodium phosphate; hydrocortisone sodium succinate; hydrocortisonevalerate; hydroxyprogesterone; inokosterone; isoflupredone;isoflupredone acetate; isoprednidene; loteprednol etabonate;meclorisone; mecortolon; medrogestone; medroxyprogesterone; medrysone;megestrol; megestrol acetate; melengestrol; meprednisone;methandrostenolone; methylprednisolone; methylprednisolone aceponate;methylprednisolone acetate; methylprednisolone hemisuccinate;methylprednisolone sodium succinate; methyltestosterone; metribolone;mometasone; mometasone furoate; mometasone furoate monohydrate; nisone;nomegestrol; norgestomet; norvinisterone; oxymesterone; paramethasone;paramethasone acetate; ponasterone; prednicarbate; prednisolamate;prednisolone; prednisolone 21-diethylaminoacetate; prednisolone21-hemisuccinate; prednisolone acetate; prednisolone farnesylate;prednisolone hemisuccinate; prednisolone-21(beta-D-glucuronide);prednisolone metasulphobenzoate; prednisolone sodium phosphate;prednisolone steaglate; prednisolone tebutate; prednisolonetetrahydrophthalate; prednisone; prednival; prednylidene; pregnenolone;procinonide; tralonide; progesterone; promegestone; rhapontisterone;rimexolone; roxibolone; rubrosterone; stizophyllin; tixocortol;topterone; triamcinolone; triamcinolone acetonide; triamcinoloneacetonide 21-palmitate; triamcinolone benetonide; triamcinolonediacetate; triamcinolone hexacetonide; trimegestone; turkesterone; andwortmannin.

Steroid Receptor Modulators

Steroid receptor modulators (e.g., antagonists and agonists) may be usedas a substitute for or in addition to a corticosteroid in the methods,compositions, and kits of the invention. Glucocorticoid receptormodulators that may used in the methods, compositions, and kits of theinvention include compounds described in U.S. Pat. Nos. 6,380,207,6,380,223, 6,448,405, 6,506,766, and 6,570,020, U.S. Patent ApplicationPublication Nos. 2003/0176478, 2003/0171585, 2003/0120081, 2003/0073703,2002/015631, 2002/0147336, 2002/0107235, 2002/0103217, and 2001/0041802,and PCT Publication No. WO00/66522, each of which is hereby incorporatedby reference. Other steroid receptor modulators may also be used in themethods, compositions, and kits of the invention are described in U.S.Pat. Nos. 6,093,821, 6,121,450, 5,994,544, 5,696,133, 5,696,127,5,693,647, 5,693,646, 5,688,810, 5,688,808, and 5,696,130, each of whichis hereby incorporated by reference.

Other Compounds

Other compounds that may be used as a substitute for or in addition to acorticosteroid in the methods, compositions, and kits of the inventionA-348441 (Karo Bio), adrenal cortex extract (GlaxoSmithKline), alsactide(Aventis), amebucort (Schering AG), amelometasone (Taisho), ATSA(Pfizer), bitolterol (Elan), CBP-2011 (InKine Pharmaceutical),cebaracetam (Novartis) CGP-13774 (Kissei), ciclesonide (Altana),ciclometasone (Aventis), clobetasone butyrate (GlaxoSmithKline),cloprednol (Hoffmann-La Roche), collismycin A (Kirin), cucurbitacin E(NIH), deflazacort (Aventis), deprodone propionate (SSP), dexamethasoneacefurate (Schering-Plough), dexamethasone linoleate (GlaxoSmithKline),dexamethasone valerate (Abbott), difluprednate (Pfizer), domoprednate(Hoffmann-La Roche), ebiratide (Aventis), etiprednol dicloacetate(IVAX), fluazacort (Vicuron), flumoxonide (Hoffmann-La Roche),fluocortin butyl (Schering AG), fluocortolone monohydrate (Schering AG),GR-250495X (GlaxoSmithKline), halometasone (Novartis), halopredone(Dainippon), HYC-141 (Fidia), icomethasone enbutate (Hovione),itrocinonide (AstraZeneca), L-6485 (Vicuron), Lipocort (Draxis Health),locicortone (Aventis), meclorisone (Schering-Plough), naflocort(Bristol-Myers Squibb), NCX-1015 (NicOx), NCX-1020 (NicOx), NCX-1022(NicOx), nicocortonide (Yamanouchi), NIK-236 (Nikken Chemicals), NS-126(SSP), Org-2766 (Akzo Nobel), Org-6632 (Akzo Nobel), P16CM,propylmesterolone (Schering AG), RGH-1113 (Gedeon Richter), rofleponide(AstraZeneca), rofleponide palmitate (AstraZeneca), RPR-106541(Aventis), RU-26559 (Aventis), Sch-19457 (Schering-Plough), T25 (MatrixTherapeutics), TBI-PAB (Sigma-Tau), ticabesone propionate (Hoffmann-LaRoche), tifluadom (Solvay), timobesone (Hoffmann-La Roche), TSC-5(Takeda), and ZK-73634 (Schering AG).

Non-Steroidal Immunophilin-Dependent Immunosuppressants

In certain embodiments, the invention features methods, compositions,and kits employing a non-steroidal immunophilin-dependentimmunosuppressant (NsIDI).

In healthy individuals the immune system uses cellular effectors, suchas B-cells and T-cells, to target infectious microbes and abnormal celltypes while leaving normal cells intact. In individuals with anautoimmune disorder or a transplanted organ, activated T-cells damagehealthy tissues. Calcineurin inhibitors (e.g., cyclosporines,tacrolimus, pimecrolimus, ABT-281, ISAtx247), and rapamycin target manytypes of immunoregulatory cells, including T-cells, and suppress theimmune response in organ transplantation and autoimmune disorders.

Cyclosporines

The cyclosporines are fungal metabolites that comprise a class of cyclicoligopeptides that act as immunosuppressants. Cyclosporine A is ahydrophobic cyclic polypeptide consisting of eleven amino acids. Itbinds and forms a complex with the intracellular receptor cyclophilin.The cyclosporine/cyclophilin complex binds to and inhibits calcineurin,a Ca²⁺-calmodulin-dependent serine-threonine-specific proteinphosphatase. Calcineurin mediates signal transduction events requiredfor T-cell activation (reviewed in Schreiber et al., Cell 70:365-368,1991). Cyclosporines and their functional and structural analogssuppress the T cell-dependent immune response by inhibitingantigen-triggered signal transduction. This inhibition decreases theexpression of proinflammatory cytokines, such as IL-2.

Many different cyclosporines (e.g., cyclosporine A, B, C, D, E, F, G, H,and I) are produced by fungi. Cyclosporine A is a commercially availableunder the trade name NEORAL from Novartis. Cyclosporine A structural andfunctional analogs include cyclosporines having one or more fluorinatedamino acids (described, e.g., in U.S. Pat. No. 5,227,467); cyclosporineshaving modified amino acids (described, e.g., in U.S. Pat. Nos.5,122,511 and 4,798,823); and deuterated cyclosporines, such as ISAtx247(described in U.S. Patent Application Publication No. 2002/0132763 A1).Additional cyclosporine analogs are described in U.S. Pat. Nos.6,136,357, 4,384,996, 5,284,826, and 5,709,797. Cyclosporine analogsinclude, but are not limited to, D-Sar (α-SMe)³ Val²-DH-Cs (209-825),Allo-Thr-2-Cs, Norvaline-2-Cs, D-Ala(3-acetylamino)-8-Cs, Thr-2-Cs, andD-MeSer-3-Cs, D-Ser(O—CH₂CH₂—OH)-8-Cs, and D-Ser-8-Cs, which aredescribed in Cruz et al. (Antimicrob. Agents Chemother. 44:143-149,2000).

Cyclosporines are highly hydrophobic and readily precipitate in thepresence of water (e.g. on contact with body fluids). Methods ofproviding cyclosporine formulations with improved bioavailability aredescribed in U.S. Pat. Nos. 4,388,307, 6,468,968, 5,051,402, 5,342,625,5,977,066, and 6,022,852. Cyclosporine microemulsion compositions aredescribed in U.S. Pat. Nos. 5,866,159, 5,916,589, 5,962,014, 5,962,017,6,007,840, and 6,024,978.

Tacrolimus

Tacrolimus (FK506) is an immunosuppressive agent that targets T cellintracellular signal transduction pathways. Tacrolimus binds to anintracellular protein FK506 binding protein (FKBP-12) that is notstructurally related to cyclophilin. The FKBP/FK506 complex binds tocalcineurin and inhibits calcineurin's phosphatase activity. Thisinhibition prevents the dephosphorylation and nuclear translocation ofnuclear factor of activated T cells (NFAT), a nuclear component thatinitiates gene transcription required for proinflammatory cytokine(e.g., IL-2, gamma interferon) production and T cell activation. Thus,tacrolimus inhibits T cell activation.

Tacrolimus is a macrolide antibiotic that is produced by Streptomycestsukubaensis. It suppresses the immune system and prolongs the survivalof transplanted organs. It is currently available in oral and injectableformulations. Tacrolimus capsules contain 0.5 mg, 1 mg, or 5 mg ofanhydrous tacrolimus within a gelatin capsule shell. The injectableformulation contains 5 mg anhydrous tacrolimus in castor oil and alcoholthat is diluted with 0.9% sodium chloride or 5% dextrose prior toinjection. While oral administration is preferred, patients unable totake oral capsules may receive injectable tacrolimus. The initial doseshould be administered no sooner than six hours after transplant bycontinuous intravenous infusion.

Tacrolimus and tacrolimus analogs are described by Tanaka et al. (J. Am.Chem. Soc., 109:5031, 1987) and in U.S. Pat. Nos. 4,894,366, 4,929,611,and 4,956,352. FK506-related compounds, including FR-900520, FR-900523,and FR-900525, are described in U.S. Pat. No. 5,254,562; O-aryl,O-alkyl, O-alkenyl, and O-alkynylmacrolides are described in U.S. Pat.Nos. 5,250,678, 532,248, 5,693,648; amino O-aryl macrolides aredescribed in U.S. Pat. No. 5,262,533; alkylidene macrolides aredescribed in U.S. Pat. No. 5,284,840; N-heteroaryl, N-alkylheteroaryl,N-alkenylheteroaryl, and N-alkynylheteroaryl macrolides are described inU.S. Pat. No. 5,208,241; aminomacrolides and derivatives thereof aredescribed in U.S. Pat. No. 5,208,228; fluoromacrolides are described inU.S. Pat. No. 5,189,042; amino O-alkyl, O-alkenyl, andO-alkynylmacrolides are described in U.S. Pat. No. 5,162,334; andhalomacrolides are described in U.S. Pat. No. 5,143,918.

Tacrolimus is extensively metabolized by the mixed-function oxidasesystem, in particular, by the cytochrome P-450 system. The primarymechanism of metabolism is demethylation and hydroxylation. Whilevarious tacrolimus metabolites are likely to exhibit immunosuppressivebiological activity, the 13-demethyl metabolite is reported to have thesame activity as tacrolimus.

Pimecrolimus

Pimecrolimus is the 33-epi-chloro derivative of the macrolactamascomyin. Pimecrolimus structural and functional analogs are describedin U.S. Pat. No. 6,384,073. Pimecrolimus is particularly useful for thetreatment of atopic dermatitis. Pimecrolimus is currently available as a1% cream.

Rapamycin

Rapamycin is a cyclic lactone produced by Streptomyces hygroscopicus.Rapamycin is an immunosuppressive agent that inhibits T cell activationand proliferation. Like cyclosporines and tacrolimus, rapamycin forms acomplex with the immunophilin FKBP-12, but the rapamycin-FKBP-12 complexdoes not inhibit calcineurin phosphatase activity. The rapamycinimmunophilin complex binds to and inhibits the mammalian kinase targetof rapamycin (mTOR). mTOR is a kinase that is required for cell-cycleprogression. Inhibition of mTOR kinase activity blocks T cell activationand proinflammatory cytokine secretion.

Rapamycin structural and functional analogs include mono- and diacylatedrapamycin derivatives (U.S. Pat. No. 4,316,885); rapamycin water-solubleprodrugs (U.S. Pat. No. 4,650,803); carboxylic acid esters (PCTPublication No. WO 92/05179); carbamates (U.S. Pat. No. 5,118,678);amide esters (U.S. Pat. No. 5,118,678); biotin esters (U.S. Pat. No.5,504,091); fluorinated esters (U.S. Pat. No. 5,100,883); acetals (U.S.Pat. No. 5,151,413); silyl ethers (U.S. Pat. No. 5,120,842); bicyclicderivatives (U.S. Pat. No. 5,120,725); rapamycin dimers (U.S. Pat. No.5,120,727); O-aryl, O-alkyl, O-alkyenyl and O-alkynyl derivatives (U.S.Pat. No. 5,258,389); and deuterated rapamycin (U.S. Pat. No. 6,503,921).Additional rapamycin analogs are described in U.S. Pat. Nos. 5,202,332and 5,169,851.

Peptide Moieties

Peptides, peptide mimetics, peptide fragments, either natural, syntheticor chemically modified, that impair the calcineurin-mediateddephosphorylation and nuclear translocation of NFAT are suitable for usein practicing the invention. Examples of peptides that act ascalcineurin inhibitors by inhibiting the NFAT activation and the NFATtranscription factor are described, e.g., by Aramburu et al., Science285:2129-2133, 1999) and Aramburu et al., Mol. Cell. 1:627-637, 1998).As a class of calcineurin inhibitors, these agents are useful in themethods, compositions, and kits of the invention.

Therapy

The invention features methods for treating an ophthalmic disorder.While the examples describe a two-drug combination, it is understoodthat the combination of multiple agents is often desirable. Additionaltherapies are described below.

Desirably, the methods, compositions, and kits of the invention are moreeffective than other methods, compositions, and kits. By “moreeffective” is meant that a method, composition, or kit exhibits greaterefficacy, is less toxic, safer, more convenient, better tolerated, orless expensive, or provides more treatment satisfaction than anothermethod, composition, or kit with which it is being compared.

Additional Compounds

Unless the intended purpose of use is affected adversely, theprophylactic and therapeutic medicament of the present invention maycontain or may be used together with other appropriate pharmacologicallyeffective substances. This includes the combination of a corticosteroidand NsIDI with other pharmacologically effective substances as well aseither a corticosteroid or NsIDI with other pharmacologically effectivesubstances. Exemplary pharmacologically effective substances aredipivefrin (e.g., dipivefrin hydrochloride ophthalmic 0.1%), anti-VEGFtherapies (e.g., bevacizumab, pegaptanib (MACUGEN) ranibizumab,anecortave acetate, and squalamine lactate), photodynamic therapy (e.g.,VISUDYNE (verteporfin)), NSAIDS (e.g., suprofen, indomethacin,flurbiprofen, ketorolac, diclofenac sodium, pranoprofen), antiallergicagents (e.g., ibudilast, tranilast, ketotifen fumarate, sodiumcromoglicate, cromolyn sodium, nedocromil sodium, azelastine),antihistamines (e.g., epinastine, emedastine, levocabastine,olopatadine, diphenhydramine hydrochloride), glaucoma-treating agents(e.g., bimatoprost, apraclonidine, travoprost, latanoprost, brimonidine,pilocarpine hydrochloride, physostigmine salicylate, timolol,isopropylunoprostone), artificial tears, antibiotics (e.g., gentamycinsulfate, fradiomycin sulfate, tobramycin, sulbenicillin, cefinenoxime,erythromycin, colistin, oxytetracycline, polymyxin B, chloramphenicol,micronomicin, dibekacin, sisomicin, sulfamethizole, sulfamethoxazole,ofloxacin, norfloxacin, lomefloxacin hydrochloride, enoxacin,ciprofloxacin hydrochloride, cinoxacin, sparfloxacin, tosufloxacintosylate, nalidixic acid, pipemidici acid trihydrate, pipemidic acid,fleroxacin, levofloxacin), antiviral agents (e.g., idoxuridine,acyclovir, ganciclovir), and antimycotic agents (e.g., pimaricin,fluconazole, miconazole, amphotericin B, flucytosine, itraconazole).

Ophthalmic Disorders

The methods, compositions, and kits of the invention may be used for thetreatment of ophthalmic disorders. Examples of ophthalmic disorders areage related macular degeneration, alkaline erosive keratoconjunctivitis,allergic conjunctivitis, allergic keratitis, anterior uveitis, Behcet'sdisease, blepharitis, blood-aqueous barrier disruption, chorioiditis,chronic uveitis, conjunctivitis, contact lens-inducedkeratoconjunctivitis, corneal abrasion, corneal trauma, corneal ulcer,crystalline retinopathy, cystoid macular edema, dacryocystitis, diabetickeratophathy, diabetic macular edema, diabetic retinopathy, dry eyedisease, dry age-related macular degeneration, eosinophilic granuloma,episcleritis, exudative macular edema, Fuchs' Dystrophy, giant cellarteritis, giant papillary conjunctivitis, glaucoma, glaucoma surgeryfailure, graft rejection, herpes zoster, inflammation after cataractsurgery, iridocorneal endothelial syndrome, iritis, keratoconjunctivasicca, keratoconjunctival inflammatory disease, keratoconus, latticedystrophy, map-dot-fingerprint dystrophy, necrotic keratitis,neovascular diseases involving the retina, uveal tract or cornea such asneovascular glaucoma, corneal neovascularization, neovascularizationresulting following a combined vitrectomy and lensectomy,neovascularization of the optic nerve, and neovascularization due topenetration of the eye or contusive ocular injury, neuroparalytickeratitis, non-infectious uveitisocular herpes, ocular lymphoma, ocularrosacea, ophthalmic infections, ophthalmic pemphigoid, optic neuritis,panuveitis, papillitis, pars planitis, persistent macular edema,phacoanaphylaxis, posterior uveitis, post-operative inflammation,proliferative diabetic retinopathy, proliferative sickle cellretinopathy, proliferative vitreoretinopathy, retinal artery occlusion,retinal detachment, retinal vein occlusion, retinitis pigmentosa,retinopathy of prematurity, rubeosis iritis, scleritis, Stevens-Johnsonsyndrome, sympathetic ophthalmia, temporal arteritis, thyroid associatedophthalmopathy, uveitis, vernal conjunctivitis, vitamin Ainsufficiency-induced keratomalacia, vitreitis, or wet age-relatedmacular degeneration.

Administration

In particular embodiments of any of the methods of the invention, thecompounds are administered within 14 days of each other, within 10 daysof each other, within five days of each other, within twenty-four hoursof each other, or simultaneously. The compounds may be formulatedtogether as a single composition, or may be formulated and administeredseparately. One or both compounds may be administered in a low dosage orin a high dosage, each of which is defined herein. It may be desirableto administer to the patient other compounds, such as a humectant, NSAID(e.g., naproxen sodium, diclofenac sodium, diclofenac potassium,aspirin, sulindac, diflunisal, piroxicam, indomethacin, ibuprofen,nabumetone, choline magnesium trisalicylate, sodium salicylate,salicylsalicylic acid, fenoprofen, flurbiprofen, ketoprofen,meclofenamate sodium, meloxicam, oxaprozin, sulindac, and tolmetin),COX-2 inhibitor (e.g., rofecoxib, celecoxib, valdecoxib, andlumiracoxib), glucocorticoid receptor modulator, or DMARD.

Therapy according to the invention may be performed alone or inconjunction with another therapy and may be provided at home, thedoctor's office, a clinic, a hospital's outpatient department, or ahospital. Treatment optionally begins at a hospital so that the doctorcan observe the therapy's effects closely and make any adjustments thatare needed, or it may begin on an outpatient basis. The duration of thetherapy depends on the type of disease or disorder being treated, theage and condition of the patient, the stage and type of the patient'sdisease, and how the patient responds to the treatment. Additionally, aperson having a greater risk of developing an ophthalmic disease (e.g.,a person who is undergoing age-related hormonal changes) may receivetreatment to inhibit or delay the onset of symptoms.

Each compound of the combination may be formulated in a variety of waysthat are known in the art. For example, the first and second agents maybe formulated together or separately. Desirably, the first and secondagents are formulated together for the simultaneous or near simultaneousadministration of the agents. Such co-formulated compositions caninclude the two drugs together in the same ointment, cream, foam,liquid, etc. By using different formulation strategies for differentagents, the pharmacokinetic profiles for each agent can be suitablymatched.

Ophthalmic Formulations

Ophthalmic formulations include but are not limited to ocular injectionssuch as intravitreal, subtenons, subconjunctival, periocular,retrobulbar injections; topical ophthalmic aqueous solutions, such assuspensions, ointments, and gels; intraocular biodegradable andnon-biodegradable implants; implants that are inserted through incisionsmade in the eye wall or sutured around the globe of the eye; tack forintraocular drug delivery; and bioadhesive ophthalmic inserts.

For topical ophthalmic administration of combinations of the invention,formulations may take the form of solutions, gels, ointments,suspensions or solid inserts, formulated so that a unit dosage comprisesa therapeutically effective amount of each active component or somesubmultiple thereof.

Typical ophthalmologically acceptable carriers are, for example, water,mixtures of water and water-miscible solvents such as lower alkanols oraralkanols, vegetable oils, polyalkylene glycols, petroleum based jelly,ethyl cellulose, ethyl oleate, carboxymethylcellulose,polyvinylpyrrolidone, isopropyl myristate and other conventionallyemployed acceptable carriers. The pharmaceutical preparation may alsocontain non-toxic auxiliary substances such as emulsifying, preserving,wetting agents, bodying agents and the like, as for example,polyethylene glycols 200, 300, 400 and 600, carbowaxes 1,000, 1,500,4,000, 6,000 and 10,000, antibacterial components such as quaternaryammonium compounds, phenylmercuric salts known to have cold sterilizingproperties and which are non-injurious in use, thimerosal, benzalkoniumchloride, methyl and propyl paraben, benzyldodecinium bromide, benzylalcohol, phenylethanol, buffering ingredients such as sodium chloride,sodium borate, sodium acetate, or gluconate buffers, and otherconventional ingredients such as sorbitan monolaurate, triethanolamine,polyoxyethylene sorbitan monopalmitylate, dioctyl sodium sulfosuccinate,monothioglycerol, thiosorbitol, ethylenediamine tetra-acetic acid, andthe like. Additionally, suitable ophthalmic vehicles can be used ascarrier media for the present purpose including conventional phosphatebuffer vehicle systems, isotonic boric acid vehicles, isotonic sodiumchloride vehicles, isotonic sodium borate vehicles and the like.

The formulation may also include a gum such as gellan gum at aconcentration of, for example, 0.1 to 2% by weight so that the aqueouseyedrops gel on contact with the eye, thus providing the advantages of asolid ophthalmic insert as described in U.S. Pat. No. 4,861,760.

The pharmaceutical preparation may also be in the form of a solid insertsuch as one which after dispensing the drug remains essentially intactas described in U.S. Pat. Nos. 4,256,108; 4,160,452; and 4,265,874; or abio-erodible insert that either is soluble in lacrimal fluids, orotherwise disintegrates as described in U.S. Pat. No. 4,287,175 or EPOpublication 0077261.

Other ophthalmic formulations and delivery devices are described in U.S.Pat. Nos. 4,014,335; 4,300,557; 5,098,443; 5,188,826; 5,378,475;5,422,116; 5,424,078; 5,466,233; 5,725,493; 5,773,019; 5,773,021;5,776,445; 5,814,635; 5,888,493; 6,235,781; 6,297,228; 6,372,245;6,511,660; 6,579,519; 6,582,422; 6,713,081; 6,719,750; and U.S. PatentApplication Publication Nos. 2002-0064513; 2003-0232089; and2005-0234018.

Kits

In general, kits of the invention contain a corticosteroid and/or anNsIDI. These compounds can be provided in the kit as separatecompositions, or combined into a single composition. The kits of theinvention can also contain instructions for the administration of boththe corticosteroid and NsIDI.

Kits of the invention can also contain instructions for administering anadditional pharmacologically acceptable substance (e.g., dipivefrin,anti-VEGF therapies, photodynamic therapy, NSAIDS, antiallergic agents,antihistamines, glaucoma-treating agents, artificial tears, antibiotics,antiviral agents, and antimycotic agents) with a corticosteroid and/oran NsIDI. This kit may contain any combination of the corticosteroid,NsIDI, and additional pharmaceutically acceptable substance (i.e., anyone, two, or three of the above compounds).

Dosages

The lowest approved concentrations for ophthalmic formulations ofcertain corticosteroids are set forth in Table 1.

TABLE 1 Lowest approved concentration for Ophthalmic ophthalmic Loweststandard corticosteroid administration recommended dosage ClocortolonePivalate  0.1% N/A Hydrocortisone  1.0%  0.5 μg/3 times dailyDexamethasone  0.1% 0.05 μg/4-6 times daily Fluorometholone  0.1% 0.05μg/2-4 times daily Loteprednol Etabonate  0.2%  0.1 μg/4 times dailyMedrysone  1.0%  0.5 μg/up to every 4 hours Prednisolone Acetate 0.12%0.06 μg/2-4 times daily Rimexolone  1.0%  0.5 μg/4 times daily (N/A =Not Available)

In general, a corticosteroid can be administered at a concentrationbetween 0.01% and 5% (e.g., 5.0%, 4.0%, 3.0%, 2.0%, 1.0%, 0.9%, 0.8%,0.7%, 0.6%, 0.5%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, and 0.01%).

Low concentrations of corticosteroids of the invention are 95% or lessof the lowest approved concentration. For example, low concentrations ofcorticosteroids of the invention can be 90%, 85%, 80%, 70%, 60%, 50%,25%, 10%, 5%, 2%, 1%, 0.5% or 0.1% of the lowest approved concentration.

For example, a low concentration of clocortolone pivalate is between0.01% and 0.1% (e.g., 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, and0.01%), a low concentration of hydrocortisone is between 0.01% and 1.0%(e.g., 1.0%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.1%, 0.09%, 0.08%, 0.07%,0.06%, 0.05%, and 0.01%), a low concentration of dexamethasone isbetween 0.01% and 0.1% (e.g., 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%,and 0.01%), a low concentration of fluorometholone is between 0.01% and0.1% (e.g., 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, and 0.01%), a lowconcentration of loteprednol etabonate is between 0.01% and 0.2% (e.g.,0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, and 0.01%), a lowconcentration of medrysone is between 0.01% and 1.0% (e.g., 1.0%, 0.9%,0.8%, 0.7%, 0.6%, 0.5%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, and0.01%), a low concentration of rimexolone is between 0.01% and 1.0%(e.g., 1.0%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.1%, 0.09%, 0.08%, 0.07%,0.06%, 0.05%, and 0.01%), and a low concentration of prednisolone isbetween 0.01% and 0.12% (e.g., 0.12%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%,0.05%, and 0.01%).

The lowest approved ophthalmic concentration of cyclosporine A is 0.05%.Low concentrations of cyclosporine A are 0.04%, or more preferably0.03%, 0.02%, 0.01%, 0.008%, 0.005%, or 0.001%. The standard dailyophthalmic dosage of cyclosporine A is 0.2 μg twice daily.

Continuous daily dosing with the combinations of the invention may notbe required. A therapeutic regimen may require cycles, during which timea drug is not administered, or therapy may be provided on an as neededbasis during periods of acute inflammation.

The compound may be administered by methods described herein (e.g., thecompound can be administered topically in the form of foams, lotions,drops, creams, ointments, emulsions, or gels).

In order to test the efficacy of the proposed low concentrations andratios of the corticosteroid and cyclosporine combination forback-of-the-eye diseases, a rabbit model of retinal vein occlusion isutilized. Since corticosteroids may modulate VEGF-mediated responses invivo, the effects of the low-concentration corticosteroid andcyclosporine combination are evaluated in a rabbit model of VEGF-inducedblood-retinal barrier and blood-aqueous (iris) barrier breakdown. VEGFinjected intravitreally induces a time and dose-dependent breakdown ofthe blood-retinal and blood-aqueous barriers. VEGF165 or vehicle isfirst injected intravitreally in female Dutch Belt rabbits, and scanningocular fluorophotometry is used to non-invasively measure thefluorescein leakage from retinal and iris vasculature. Subsequently, therabbits are treated with either the low-concentration corticosteroid andcyclosporine or vehicle (s.c. or intravitreal) to determine its efficacyon inhibiting the blood-retinal barrier and blood-aqueous breakdown. Theeffects of corticosteroid and cyclosporine or vehicle on VEGF-inducedretinal vasculopathy are further assessed with fundus imaging,fluorescein angiography, and ocular coherence tomography. Additionalmethods for determining the low concentrations of the invention for thetreatment of back-of-the-eye diseases are set forth in Qui et al., ExpEye Res. 83:141 2006; Manzano et al., Retina. 26:257 2006; Edelman etal., Exp Eye Res. 80:249 2005; Arroyo et al., Jpn J. Ophthalmol. 45:3592001; Ieki et al., Curr Eye Res. 25:317 2002; and Takei et al., GraefesArch Clin Exp Ophthalmol. 231:476 1993, each of which is herebyincorporated by reference in its entirety.

In order to test the efficacy the proposed low concentrations and ratiosof the corticosteroid and cyclosporine combination for front-of-the-eyediseases a murine model of keratoconjunctivitis sicca is used. In oneexample, dry eye is induced in mice by subcutaneous injection ofscopolamine and by exposing them to an air draft and low-humidityenvironment for 12 days. After 12 days, the mice are killed, and theeyes and eyelids are excised, frozen, and cryosectioned. Transmissionelectron microscopy (TEM) is performed on conjunctival and cornealsamples are taken from the eyes. The effect of low-concentrationcorticosteroid and cyclosporine on apoptosis is detected in frozensections with the ApopTag (ISOL) In Situ Oligo Ligation Kit, whichspecifically detects DNA fragmentation. Immunohistochemical staining isperformed to detect activated caspase-3. Conjunctival goblet cell numberis counted in tissue sections stained with period acid Schiff (PAS)reagent. These assays are used to determine the effects oflow-concentration corticosteroid and cyclosporine on its ability toreduce conjunctival epithelial apoptosis and protect against goblet cellloss. Additional methods for determining the low concentrations fortreating front-of-the-eye diseases are set forth in Strong et al.Cornea. 24:80 2005; Luo et al., Invest Ophthalmol V is Sci. 45:42932004; Yeh et al., Invest Ophthalmol V is Sci. 44:124 2003; andPflugfelder et al., Am J. Pathol. 166:61 2005, each of which is herebyincorporated by reference in its entirety.

Additional Applications

The compounds of the invention can be employed in immunomodulatory ormechanistic assays to determine whether other combinations, or singleagents, are as effective as the combination in inhibiting secretion orproduction of proinflammatory cytokines or modulating immune responseusing assays generally known in the art, examples of which are describedherein. After a suitable time, the cells are examined for cytokinesecretion or production or other suitable immune response. The relativeeffects of the combinations versus each other, and versus the singleagents are compared, and effective compounds and combinations areidentified.

The combinations of the invention are also useful tools in elucidatingmechanistic information about the biological pathways involved ininflammation. Such information can lead to the development of newcombinations or single agents for inhibiting inflammation caused byproinflammatory cytokines. Methods known in the art to determinebiological pathways can be used to determine the pathway, or network ofpathways affected by contacting cells stimulated to produceproinflammatory cytokines with the compounds of the invention. Suchmethods can include, analyzing cellular constituents that are expressedor repressed after contact with the compounds of the invention ascompared to untreated, positive or negative control compounds, and/ornew single agents and combinations, or analyzing some other metabolicactivity of the cell such as enzyme activity, nutrient uptake, andproliferation. Cellular components analyzed can include genetranscripts, and protein expression. Suitable methods can includestandard biochemistry techniques, radiolabeling the compounds of theinvention (e.g., ¹⁴C or ³H labeling), and observing the compoundsbinding to proteins, e.g. using 2d gels, gene expression profiling. Onceidentified, such compounds can be used in in vivo models to furthervalidate the tool or develop new anti-inflammatory agents.

Experimental Results

Both corticosteroids and NsIDIs suppress cytokine production in cellculture models of immune function. We tested the effect of thecombination of various concentrations of NsIDIs and corticosteroids oncytokine production in a cell culture model of immune function. Wepropose that combinations that demonstrate synergistic or superaddativeeffects can be used to treat ophthalmic disorders at concentrations lowenough to avoid undesired side effects.

Assay for Proinflammatory Cytokine-Suppressing Activity

Compound dilution matrices were assayed for the suppression of IFNγ,IL-2, and TNFα, as described below. The results from these experimentsare set forth in FIGS. 1A-1LL.

IFNγ

A 100 μL suspension of diluted human white blood cells contained withineach well of a polystyrene 384-well plate (NalgeNunc) was stimulated tosecrete IFNγ by treatment with a final concentration of 10 ng/mL phorbol12-myristate 13-acetate (Sigma, P-1585) and 750 ng/mL ionomycin (Sigma,1-0634). Various concentrations of each test compound were added at thetime of stimulation. After 16-18 hours of incubation at 37° C. in ahumidified incubator, the plate was centrifuged and the supernatanttransferred to a white opaque polystyrene 384 well plate (NalgeNunc,Maxisorb) coated with an anti-IFNγ antibody (Endogen, #M-700A-E). Aftera two-hour incubation, the plate was washed (Tecan PowerWasher 384) withphosphate buffered saline (PBS) containing 0.1% Tween 20(polyoxyethylene sorbitan monolaurate) and incubated for an additionalone hour with another anti-IFNγ antibody that was biotin labeled(Endogen, M701B) and horseradish peroxidase (HRP) coupled to strepavidin(PharMingen, #13047E). After the plate was washed with 0.1% Tween20/PBS, an HRP-luminescent substrate was added to each well and lightintensity measured using a LJL Analyst plate luminometer.

IL-2

A 100 μL suspension of diluted human white blood cells contained withineach well of a polystyrene 384-well plate (NalgeNunc) was stimulated tosecrete IL-2 by treatment with a final concentration of 10 ng/mL phorbol12-myristate 13-acetate (Sigma, P-1585) and 750 ng/mL ionomycin (Sigma,1-0634). Various concentrations of each test compound were added at thetime of stimulation. After 16-18 hours of incubation at 37° C. in ahumidified incubator, the plate was centrifuged and the supernatanttransferred to a white opaque polystyrene 384 well plate (NalgeNunc,Maxisorb) coated with an anti-IL-2 antibody (PharMingen, #555051). Aftera two-hour incubation, the plate was washed (Tecan PowerWasher 384) withPBS containing 0.1% Tween 20 and incubated for an additional one hourwith another anti-IL-2 antibody that was biotin labeled (Endogen, M600B)and HRP coupled to strepavidin (PharMingen, #13047E). After the platewas washed with 0.1% Tween 20/PBS, an HRP-luminescent substrate wasadded to each well and light intensity measured using a LJL Analystplate luminometer.

TNFα

The effects of test compound combinations on TNFα secretion were assayedin white blood cells from human buffy coat stimulated with phorbol12-myistate 13-acetate as follows. Human white blood cells from buffycoat were diluted 1:50 in media (RPMI; Gibco BRL, #11875-085), 10% fetalbovine serum (Gibco BRL, #25140-097), 2% penicillin/streptomycin (GibcoBRL, #15140-122)) and 50 μL of the diluted white blood cells was placedin each well of the assay plate. Drugs were added to the indicatedconcentration. After 16-18 hours of incubation at 37° C. with 5% CO₂ ina humidified incubator, the plate was centrifuged and the supernatanttransferred to a white opaque polystyrene 384-well plate (NalgeNunc,Maxisorb) coated with an anti-TNFα antibody (PharMingen, #551220). Aftera two-hour incubation, the plate was washed (Tecan Powerwasher 384) withPBS containing 0.1% Tween 20 and incubated for one additional hour withbiotin labeled anti-TNFα antibody (PharMingen, #554511) and HRP coupledto streptavidin (PharMingen, #13047E). The plate was then washed againwith 0.1% Tween 20/PBS. An HRP-luminescent substrate was added to eachwell, and the light intensity of each well was measured using a plateluminometer.

Percent Inhibition

The percent inhibition (% I) for each well was calculated using thefollowing formula:% I=[(avg. untreated wells−treated well)/(avg. untreated wells)]×100The average untreated well value (avg. untreated wells) is thearithmetic mean of 40 wells from the same assay plate treated withvehicle alone. Negative inhibition values result from local variationsin treated wells as compared to untreated wells.Preparation of Compounds

The stock solution containing cyclosporin A was made at a concentrationof 1.2 mg/ml in DMSO. The stock solution of tacrolimus was made at aconcentration of 0.04 mg/ml in DMSO. Stock solutions containing acorticosteroid were made in dimethylsulfoxide (DMSO) at a finalconcentration of between 0 and 40 μM. Master plates were prepared tocontain dilutions of the stock solutions of the compounds describedabove. Master plates were sealed and stored at −20° C. until ready foruse.

The final single agent plates were generated by transferring 1 μL ofstock solution from the specific master plate to a dilution platecontaining 100 μL of media (RPMI; Gibco BRL, #11875-085), 10% fetalbovine serum (Gibco BRL, #25140-097), 2% Penicillin/Streptomycin (GibcoBRL, #15140-122)) using the Packard Mini-Trak liquid handler. Thisdilution plate was then mixed and a 5 mL aliquot transferred to thefinal assay plate, which had been pre-filled with 50 mL/well RPMI mediacontaining the appropriate stimulant to activate IFNγ, IL-2, or TNFαsecretion.

Other Embodiments

Various modifications and variations of the described methods andcompositions of the invention will be apparent to those skilled in theart without departing from the scope and spirit of the invention.Although the invention has been described in connection with specificdesired embodiments, it should be understood that the invention asclaimed should not be unduly limited to such specific embodiments.Indeed, various modifications of the described modes for carrying outthe invention that are obvious to those skilled in the fields ofmedicine, immunology, pharmacology, endocrinology, or related fields areintended to be within the scope of the invention.

All publications mentioned in this specification are herein incorporatedby reference to the same extent as if each independent publication wasspecifically and individually incorporated by reference.

1. A method of treating an ophthalmic disorder in a patient, said methodcomprising ophthalmically administering prednisolone or prednisoloneacetate and cyclosporine A, wherein prednisolone or prednisolone acetateis administered at a concentration of 0.12% and said cyclosporine A isadministered at a concentration of 0.01% or 0.02% wherein said disorderis selected from the group consisting of allergic conjunctivitis and dryeye.
 2. The method of claim 1, wherein said ophthalmic disorder is dryeye.
 3. The method of claim 1, wherein said ophthalmic disorder isallergic conjunctivitis.
 4. The method of claim 1, wherein saidcyclosporine A is administered at a concentration of 0.01%.
 5. Themethod of claim 1, wherein said cyclosporine A is administered at aconcentration of 0.02%.
 6. The method of claim 1, wherein saidprednisolone or prednisolone acetate and said cyclosporine A areadministered as a single composition.
 7. The method of claim 6, whereinsaid composition is a solution, gel, ointment, suspension, emulsion, orsolid insert.
 8. The method of claim 6, wherein said prednisolone orprednisolone acetate and said cyclosporine A are the sole activeingredients of said composition.